Use of sdf-1 to mitigate scar formation

ABSTRACT

The subject matter provided herein relates to method for inhibiting or mitigating scar formation in a wound of the skin, by increasing the concentration of SDF-1 in, or proximate to, the wound. As described herein SDF-1 protein or an SDF-1 expression vector can be administered to a wound or the area proximate a wound by providing a therapeutically effective amount of SDF-1 protein or an SDF-1 expression vector.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/650,726, filed Oct. 12, 2012, now allowed, which is a division ofU.S. application Ser. No. 12/808,056, filed Jun. 14, 2010, nowabandoned, which is the U.S. national stage application of InternationalApplication No. PCT/US2008/086820, filed Dec. 15, 2008, which claimspriority from U.S. Provisional Application No. 61/013,878, filed Dec.14, 2007; the subject matter of these applications is incorporated byreference herein in their entirety.

TECHNICAL FIELD

The present invention relates to composition and methods of promotingwound healing in subject.

BACKGROUND

Wounds (i.e., lacerations or openings) in mammalian tissue result intissue disruption and coagulation of the microvasculature at the woundface. Repair of such tissue represents an orderly, controlled cellularresponse to injury. All soft tissue wounds, regardless of size heal in asimilar manner. Tissue growth and repair are biologic systems whereincellular proliferation and angiogenesis occur in the presence of anoxygen gradient. The sequential morphological and structural changeswhich occur during tissue repair have been characterized in great detailand have in some instances been quantified (Hunt, T. K., et al.,“Coagulation and macrophage stimulation of angiogenesis and woundhealing,” in The Surgical Wound, pp. 1-18, ed. F. Dineen & G.Hildrick-Smith (Lea & Febiger, Philadelphia: 1981)].

The cellular morphology consists of three distinct zones. The centralavascular wound space is oxygen deficient, acidotic and hypercarbic, andhas high lactate levels. Adjacent to the wound space is a gradient zoneof local anemia (ischemia) which is populated by dividing fibroblasts.Behind the leading zone is an area of active collagen synthesischaracterized by mature fibroblasts and numerous newly-formedcapillaries (i.e., neovascularization). While this new blood vesselgrowth (angiogenesis) is necessary for the healing of wound tissue,angiogenic agents generally are unable to fulfill the long-felt need ofproviding the additional biosynthetic effects of tissue repair. Despitethe need for more rapid healing of wounds (i.e., severe burns, surgicalincisions, lacerations and other trauma), to date there has been onlylimited success in accelerating wound healing with pharmacologicalagents.

SUMMARY

The present invention relates to methods and composition of treatingand/or promoting wound healing in a subject. In the method, SDF-1 isadministered directly to the wound or cells proximate the wound at anamount effective to promote wound healing. The wound can include anyinjury to any portion of the body of a subject. Examples of wounds thatcan be treated by the method include acute conditions or wounds; such asthermal burns, chemical burns, radiation burns, burns caused by excessexposure to ultraviolet radiation (e.g., sunburn); damage to bodilytissues, such as the perineum as a result of labor and childbirth;injuries sustained during medical procedures, such as episiotomies,trauma-induced injuries including cuts, incisions, excoriations;injuries sustained from accidents; post-surgical injuries, as well aschronic conditions; such as pressure sores, bedsores, conditions relatedto diabetes and poor circulation, and all types of acne. In addition,the wound can include dermatitis, such as impetigo, intertrigo,folliculitis and eczema, wounds following dental surgery; periodontaldisease; wounds following trauma; and tumor associated wounds.

In an aspect of the invention, an amount of SDF-1 administered to thewound or cells proximate the wound can be an amount effective to promoteor accelerate wound closure and wound healing, mitigate scar formationof and/or around the wound, inhibit apoptosis of cells surrounding orproximate the wound, and/or facilitate revascularization of the woundedtissue. The SDF-1 can be administered to cells proximate the wound thatinclude SDF-1 receptors that are up-regulated as a result of tissueinjury and/or trauma. In an aspect of the invention, the SDF-1 receptorcan comprise CXCR4 and/or CXCR7, and the SDF-1 can be administered at anamount effect to increase Akt-phosphorylation of the cells.

In another aspect of the invention, the SDF-1 can be administered byexpressing SDF-1 in cells proximate the wound and/or providing apharmaceutical composition to the wound which includes SDF-1. The SDF-1can be expressed from the cells proximate the wound by geneticallymodifying the cells by at least one of a vector, plasmid DNA,electroporation, and nanoparticles to express SDF-1.

The present invention also relates to methods and composition ofinhibiting scar formation during wound healing in a subject. In themethod, SDF-1 is administered directly to the wound or cells proximatethe wound at an amount effective to mitigate scar formation in and/oraround the wound. The wound can include any injury to any portion of thebody of a subject. Examples of wound that can be treated by the methodinclude acute conditions or wounds; such as thermal burns, chemicalburns, radiation burns, burns caused by excess exposure to ultravioletradiation (e.g., sunburn); damage to bodily tissues, such as theperineum as a result of labor and childbirth; injuries sustained duringmedical procedures, such as episiotomies, trauma-induced injuriesincluding cuts, incisions, excoriations; injuries sustained fromaccidents; post-surgical injuries, as well as chronic conditions; suchas pressure sores, bedsores, conditions related to diabetes and poorcirculation, and all types of acne. In addition, the wound can includedermatitis such as impetigo, intertrigo, folliculitis and eczema, woundsfollowing dental surgery; periodontal disease; wounds following trauma;and tumor associated wounds.

In an aspect of the invention, an amount of SDF-1 administered to thewound or cells proximate the wound can be an amount effective to promoteor accelerate wound closure and wound healing, mitigate scar fibrosis ofthe tissue of and/or around the wound, inhibit apoptosis of cellssurrounding or proximate the wound, and/or facilitate revascularizationof the wounded tissue. The SDF-1 can be administered to cells proximatethe wound that include SDF-1 receptors that are up-regulated as a resultof tissue injury and/or trauma. In an aspect of the invention, the SDF-1receptor can comprise CXCR4 and/or CXCR7, and the SDF-1 can beadministered at an amount effect to increase Akt-phosphorylation of thecells.

In another aspect of the invention, the SDF-1 can be administered byexpressing SDF-1 in cells proximate the wound and/or providing apharmaceutical composition to the wound which includes SDF-1. The SDF-1can be expressed from the cells proximate the wound by geneticallymodifying the cells by at least one of a vector, plasmid DNA,electroporation, and nanoparticles to express SDF-1.

The present invention further relates to methods and composition ofpromoting or accelerating wound closure in a subject. In the method,SDF-1 is administered directly to the wound or cells proximate the woundat an amount effective to promote wound closure. The wound can includeany injury to any portion of the body of a subject. Examples of woundthat can be treated by the method include acute conditions or wounds;such as thermal burns, chemical burns, radiation burns, burns caused byexcess exposure to ultraviolet radiation (e.g., sunburn); damage tobodily tissues, such as the perineum as a result of labor andchildbirth; injuries sustained during medical procedures, such asepisiotomies, trauma-induced injuries including cuts, incisions,excoriations; injuries sustained from accidents; post-surgical injuries,as well as chronic conditions; such as pressure sores, bedsores,conditions related to diabetes and poor circulation, and all types ofacne. In addition, the wound can include dermatitis such as impetigo,intertrigo, folliculitis and eczema, wounds following dental surgery;periodontal disease; wounds following trauma; and tumor associatedwounds.

In an aspect of the invention, an amount of SDF-1 administered to thewound or cells proximate the wound can be an amount effective to promoteor accelerate wound closure and wound healing, mitigate scar formationof and/or around the wound, inhibit apoptosis of cells surrounding orproximate the wound, and/or facilitate revascularization of the woundedtissue. The SDF-1 can be administered to cells proximate the wound thatinclude SDF-1 receptors that are up-regulated as a result of tissueinjury and/or trauma. In an aspect of the invention, the SDF-1 receptorcan comprise CXCR4 and/or CXCR7, and the SDF-1 can be administered at anamount effect to increase Akt-phosphorylation of the cells.

In another aspect of the invention, the SDF-1 can be administered byexpressing SDF-1 in cells proximate the wound and/or providing apharmaceutical composition to the wound which includes SDF-1. The SDF-1can be expressed from the cells proximate the wound by geneticallymodifying the cells by at least one of a vector, plasmid DNA,electroporation, and nanoparticles to express SDF-1.

The present invention still further relates to a topical and/or localformulation for promoting wound healing in subject. The formulation caninclude an amount of SDF-1 effective to promote wound closure andinhibit scarring of the wound when the formulation is administered tothe wound.

The wound can include any injury to any portion of the body of asubject. Examples of wound that can be treated by the method includeacute conditions or wounds; such as thermal burns, chemical burns,radiation burns, burns caused by excess exposure to ultravioletradiation (e.g., sunburn); damage to bodily tissues, such as theperineum as a result of labor and childbirth; injuries sustained duringmedical procedures, such as episiotomies, trauma-induced injuriesincluding cuts, incisions, excoriations; injuries sustained fromaccidents; post-surgical injuries, as well as chronic conditions; suchas pressure sores, bedsores, conditions related to diabetes and poorcirculation, and all types of acne. In addition, the wound can includedermatitis such as impetigo, intertrigo, folliculitis and eczema, woundsfollowing dental surgery; periodontal disease; wounds following trauma;and tumor associated wounds.

The amount of SDF-1 in the wound can also be an amount effective topromote or accelerate wound healing, mitigate scar formation of and/oraround the wound, inhibit apoptosis of cells surrounding or proximatethe wound, and/or facilitate revascularization of the wounded tissue. Inan aspect of the invention, the SDF-1 can be in the form of protein orplasmid that when administered to a cell proximate the wound promotesexpression of SDF-1 from the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features of the present invention will becomeapparent to those skilled in the art to which the present inventionrelates upon reading the following description with reference to theaccompanying drawings.

FIG. 1 illustrates photographs showing that SDF-1 releasing scaffoldsaccelerate wound healing.

FIG. 2 illustrates plots showing the % Healing over a period days forporcine wounds treated with SDF-1 protein scaffold, SDF-1 plasmascaffold, Saline scaffold, and no scaffold.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. Commonly understood definitions ofmolecular biology terms can be found in, for example, Rieger et al.,Glossary of Genetics: Classical and Molecular, 5th edition,Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford UniversityPress: New York, 1994.

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises, such as Molecular Cloning:A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates). Methodsfor chemical synthesis of nucleic acids are discussed, for example, inBeaucage and Carruthers, Tetra. Letts. 22:1859-1862, 1981, and Matteucciet al., J. Am. Chem. Soc. 103:3185, 1981. Chemical synthesis of nucleicacids can be performed, for example, on commercial automatedoligonucleotide synthesizers. Immunological methods (e.g., preparationof antigen-specific antibodies, immunoprecipitation, and immunoblotting)are described, e.g., in Current Protocols in Immunology, ed. Coligan etal., John Wiley & Sons, New York, 1991; and Methods of ImmunologicalAnalysis, ed. Masseyeff et al., John Wiley & Sons, New York, 1992.Conventional methods of gene transfer and gene therapy can also beadapted for use in the present invention. See, e.g., Gene Therapy:Principles and Applications, ed. T. Blackenstein, Springer Verlag, 1999;Gene Therapy Protocols (Methods in Molecular Medicine), ed. P. D.Robbins, Humana Press, 1997; and Retro-vectors for Human Gene Therapy,ed. C. P. Hodgson, Springer Verlag, 1996.

The present invention relates to the treatment of a wound and/or thepromotion of wound healing or wound closure in a mammalian subject byadministering to the wound and/or cells proximate the wound an amount ofSDF-1 effective to promote wound healing, mitigate cell apoptosis,and/or mitigate or inhibit scar formation in the wound. The presentinvention also relates to a method of inhibiting scar formation and/orfibrosis of a wound or tissue proximate a wound by administering to thewound and/or cells or tissue proximate the wound an amount of SDF-1effective to promote wound healing, mitigate cell apoptosis, and/ormitigate or inhibit scar formation in the wound. The present inventionfurther relates to a topical and/or local formulation for treating awound comprising SDF-1 or an agent that upregulates expression of SDF-1in cells of a wound.

The wound treated by the method and/or compositions of the presentinvention can include any injury to any portion of the body of a subject(e.g., internal wound or external wound) including: acute conditions orwounds, such as thermal burns, chemical burns, radiation burns, burnscaused by excess exposure to ultraviolet radiation (e.g., sunburn);damage to bodily tissues, such as the perineum as a result of labor andchildbirth; injuries sustained during medical procedures, such asepisiotomies; trauma-induced injuries, such as cuts, incisions,excoriations, injuries sustained as result of accidents, ulcers, such aspressure ulcers, diabetic ulcers, plaster ulcers, and decubitus ulcer,post-surgical injuries. The wound can also include chronic conditions orwounds, such as pressure sores, bedsores, conditions related to diabetesand poor circulation, and all types of acne. In addition, the wound caninclude dermatitis, such as impetigo, intertrigo, folliculitis andeczema, wounds following dental surgery; periodontal disease; tumorassociated wounds.

It will be appreciated that the present application is not limited tothe preceding wounds or injuries and that other wounds or tissueinjuries whether acute and/or chronic can be treated by the compositionsand methods of the present invention.

As used herein, the term “promoting wound healing” or “promoting healingof a wound” mean augmenting, improving, increasing, or inducing closure,healing, or repair of a wound.

As used herein, the terms “treating” and “treatment” refer to reductionin severity and/or frequency of symptoms, elimination of symptoms and/orunderlying cause, prevention of the occurrence of symptoms and/or theirunderlying cause, and improvement or remediation of damage. Thus, forexample, “treating” of a wound includes increasing healing at a woundsite, promoting wound closure, and decreasing scarring of the wound.

Mammalian subjects, which will be treated by methods and compositions ofthe present invention, can include any mammal, such as human beings,rats, mice, cats, dogs, goats, sheep, horses, monkeys, apes, rabbits,cattle, etc. The mammalian subject can be in any stage of developmentincluding adults, young animals, and neonates. Mammalian subjects canalso include those in a fetal stage of development.

In accordance with an aspect of the invention, the SDF-1 can beadministered to cells proximate the wound to mitigate apoptosis of thecells and promote wound healing, promote wound closure, and/or mitigatescar formation of and/or around the wound. The cells include cells thatexpress SDF-1 receptors, which are upregulated as a result of traumaand/or tissue injury. The up-regulated SDF-1 receptors can include, forexample, CXCR4 and/or CXCR7. It was found that sustained localizedadministration of SDF-1 to cells with up-regulated SDF-1 receptors as aresult of tissue injury increases Akt phosphorylation in the cells whichin turn can mitigate apoptosis of the cells. Additionally, long-termlocalized administration of SDF-1 to tissue facilitates recruitment ofstem cells and/or progenitor cells, such as endothelial progenitorcells, expressing CXCR4 and/or CXCR7 to the site of the wound beingtreated, which can facilitate revascularization of the tissuesurrounding and/or proximate the wound.

In one example, the period of time that the SDF-1 is administered to thecells of the wound and/or proximate the wound can comprise from aboutonset of the wound and/or tissue injury to about days, weeks, or monthsafter tissue injury. It was found that topical and/or local SDF-1delivery by protein or plasmid to wounds was sufficient to increase therate of healing and wound closure. Moreover, the SDF-1 treated woundstended to have less fibrosis than non-SDF-1 treated wounds, whichsuggests SDF-1 can mitigate scarring in treated wounds. It was alsofound that immediately after onset of tissue injury, cells in the woundtissue or about the periphery or the border of the wound up regulateexpression of SDF-1. After about 24 hours, SDF-1 expression by the cellsis reduced. The SDF-1 can be administered after the SDF-1 is reduced tomitigate apoptosis of the cells.

SDF-1 in accordance with the present invention can have an amino acidsequence that is substantially similar to a native mammalian SDF-1 aminoacid sequence. The amino acid sequence of a number of differentmammalian SDF-1 protein are known including human, mouse, and rat. Thehuman and rat SDF-1 amino acid sequences are about 92% identical. SDF-1can comprise two isoforms, SDF-1 alpha and SDF-1 beta, both of which arereferred to herein as SDF-1 unless identified otherwise.

The SDF-1 can have an amino acid sequence substantially identical to SEQID NO: 1. The SDF-1 that is over-expressed can also have an amino acidsequence substantially similar to one of the foregoing mammalian SDF-1proteins. For example, the SDF-1 that is over-expressed can have anamino acid sequence substantially similar to SEQ ID NO: 2. SEQ ID NO: 2,which substantially comprises SEQ ID NO: 1, is the amino acid sequencefor human SDF-1 and is identified by GenBank Accession No. NP954637. TheSDF-1 that is over-expressed can also have an amino acid sequence thatis substantially identical to SEQ ID NO: 3. SEQ ID NO: 3 includes theamino acid sequences for rat SDF and is identified by GenBank AccessionNo. AAF01066.

The SDF-1 in accordance with the present invention can also be a variantof mammalian SDF-1, such as a fragment, analog and derivative ofmammalian SDF-1. Such variants include, for example, a polypeptideencoded by a naturally occurring allelic variant of native SDF-1 gene(i.e., a naturally occurring nucleic acid that encodes a naturallyoccurring mammalian SDF-1 polypeptide), a polypeptide encoded by analternative splice form of a native SDF-1 gene, a polypeptide encoded bya homolog or ortholog of a native SDF-1 gene, and a polypeptide encodedby a non-naturally occurring variant of a native SDF-1 gene.

SDF-1 variants have a peptide sequence that differs from a native SDF-1polypeptide in one or more amino acids. The peptide sequence of suchvariants can feature a deletion, addition, or substitution of one ormore amino acids of a SDF-1 variant. Amino acid insertions arepreferably of about 1 to 4 contiguous amino acids, and deletions arepreferably of about 1 to 10 contiguous amino acids. Variant SDF-1polypeptides substantially maintain a native SDF-1 functional activity.Examples of SDF-1 polypeptide variants can be made by expressing nucleicacid molecules within the invention that feature silent or conservativechanges. One example of an SDF-1 variant is listed in U.S. Pat. No.7,405,195, which is herein incorporated by reference in its entirety.

SDF-1 polypeptide fragments corresponding to one or more particularmotifs and/or domains or to arbitrary sizes, are within the scope of thepresent invention. Isolated peptidyl portions of SDF-1 can be obtainedby screening peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. For example, anSDF-1 polypeptides of the present invention may be arbitrarily dividedinto fragments of desired length with no overlap of the fragments, orpreferably divided into overlapping fragments of a desired length. Thefragments can be produced recombinantly and tested to identify thosepeptidyl fragments, which can function as agonists of native CXCR-4polypeptides.

Variants of SDF-1 polypeptides can also include recombinant forms of theSDF-1 polypeptides. Recombinant polypeptides preferred by the presentinvention, in addition to SDF-1 polypeptides, are encoded by a nucleicacid that can have at least 70% sequence identity with the nucleic acidsequence of a gene encoding a mammalian SDF-1.

SDF-1 variants can include agonistic forms of the protein thatconstitutively express the functional activities of native SDF-1. OtherSDF-1 variants can include those that are resistant to proteolyticcleavage, as for example, due to mutations, which alter protease targetsequences. Whether a change in the amino acid sequence of a peptideresults in a variant having one or more functional activities of anative SDF-1 can be readily determined by testing the variant for anative SDF-1 functional activity.

The SDF-1 nucleic acid that encodes the SDF-1 protein can be a native ornon-native nucleic acid and be in the form of RNA or in the form of DNA(e.g., cDNA, genomic DNA, and synthetic DNA). The DNA can bedouble-stranded or single-stranded, and if single-stranded may be thecoding (sense) strand or non-coding (anti-sense) strand. The nucleicacid coding sequence that encodes SDF-1 may be substantially similar toa nucleotide sequence of the SDF-1 gene, such as nucleotide sequenceshown in SEQ ID NO: 4 and SEQ ID NO: 5. SEQ ID NO: 4 and SEQ ID NO: 5comprise, respectively, the nucleic acid sequences for human SDF-1 andrat SDF-1 and are substantially similar to the nucleic sequences ofGenBank Accession No. NM199168 and GenBank Accession No. AF189724. Thenucleic acid coding sequence for SDF-1 can also be a different codingsequence which, as a result of the redundancy or degeneracy of thegenetic code, encodes the same polypeptide as SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO: 3.

Other nucleic acid molecules that encode SDF-1 within the invention arevariants of a native SDF-1, such as those that encode fragments, analogsand derivatives of native SDF-1. Such variants may be, for example, anaturally occurring allelic variant of a native SDF-1 gene, a homolog orortholog of a native SDF-1 gene, or a non-naturally occurring variant ofa native SDF-1 gene. These variants have a nucleotide sequence thatdiffers from a native SDF-1 gene in one or more bases. For example, thenucleotide sequence of such variants can feature a deletion, addition,or substitution of one or more nucleotides of a native SDF-1 gene.Nucleic acid insertions are preferably of about 1 to 10 contiguousnucleotides, and deletions are preferably of about 1 to 10 contiguousnucleotides.

In other applications, variant SDF-1 displaying substantial changes instructure can be generated by making nucleotide substitutions that causeless than conservative changes in the encoded polypeptide. Examples ofsuch nucleotide substitutions are those that cause changes in (a) thestructure of the polypeptide backbone; (b) the charge or hydrophobicityof the polypeptide; or (c) the bulk of an amino acid side chain.Nucleotide substitutions generally expected to produce the greatestchanges in protein properties are those that cause non-conservativechanges in codons. Examples of codon changes that are likely to causemajor changes in protein structure are those that cause substitution of(a) a hydrophilic residue (e.g., serine or threonine), for (or by) ahydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine oralanine); (b) a cysteine or proline for (or by) any other residue; (c) aresidue having an electropositive side chain (e.g., lysine, arginine, orhistidine), for (or by) an electronegative residue (e.g., glutamine oraspartine); or (d) a residue having a bulky side chain (e.g.,phenylalanine), for (or by) one not having a side chain, (e.g.,glycine).

Naturally occurring allelic variants of a native SDF-1 gene within theinvention are nucleic acids isolated from mammalian tissue that have atleast 70% sequence identity with a native SDF-1 gene, and encodepolypeptides having structural similarity to a native SDF-1 polypeptide.Homologs of a native SDF-1 gene within the invention are nucleic acidsisolated from other species that have at least 70% sequence identitywith the native gene, and encode polypeptides having structuralsimilarity to a native SDF-1 polypeptide. Public and/or proprietarynucleic acid databases can be searched to identify other nucleic acidmolecules having a high percent (e.g., 70% or more) sequence identity toa native SDF-1 gene.

Non-naturally occurring SDF-1 gene variants are nucleic acids that donot occur in nature (e.g., are made by the hand of man), have at least70% sequence identity with a native SDF-1 gene, and encode polypeptideshaving structural similarity to a native SDF-1 polypeptide. Examples ofnon-naturally occurring SDF-1 gene variants are those that encode afragment of a native SDF-1 protein, those that hybridize to a nativeSDF-1 gene or a complement of to a native SDF-1 gene under stringentconditions, and those that share at least 65% sequence identity with anative SDF-1 gene or a complement of a native SDF-1 gene.

Nucleic acids encoding fragments of a native SDF-1 gene within theinvention are those that encode, amino acid residues of native SDF-1.Shorter oligonucleotides that encode or hybridize with nucleic acidsthat encode fragments of native SDF-1 can be used as probes, primers, orantisense molecules. Longer polynucleotides that encode or hybridizewith nucleic acids that encode fragments of a native SDF-1 can also beused in various aspects of the invention. Nucleic acids encodingfragments of a native SDF-1 can be made by enzymatic digestion (e.g.,using a restriction enzyme) or chemical degradation of the full-lengthnative SDF-1 gene or variants thereof.

Nucleic acids that hybridize under stringent conditions to one of theforegoing nucleic acids can also be used in the invention. For example,such nucleic acids can be those that hybridize to one of the foregoingnucleic acids under low stringency conditions, moderate stringencyconditions, or high stringency conditions are within the invention.

Nucleic acid molecules encoding a SDF-1 fusion protein may also be usedin the invention. Such nucleic acids can be made by preparing aconstruct (e.g., an expression vector) that expresses a SDF-1 fusionprotein when introduced into a suitable target cell. For example, such aconstruct can be made by ligating a first polynucleotide encoding aSDF-1 protein fused in frame with a second polynucleotide encodinganother protein such that expression of the construct in a suitableexpression system yields a fusion protein.

The nucleic acids encoding SDF-1 can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The nucleic acids within theinvention may additionally include other appended groups such aspeptides (e.g., for targeting target cell receptors in vivo), or agentsfacilitating transport across the cell membrane, hybridization-triggeredcleavage. To this end, the nucleic acids may be conjugated to anothermolecule, (e.g., a peptide), hybridization triggered cross-linkingagent, transport agent, hybridization-triggered cleavage agent, etc.

The SDF-1 can be administered directly to the wound, about the peripheryof the wound or to cells proximate, the wound in order to mitigateapoptosis of cells proximate the wound and facilitate angiogenesis tothe wounded area as well as accelerate wound closure and inhibitscarring of the wound. The SDF-1 can be delivered to the wound or cellsproximate the wound by administering an SDF-1 protein to the wound orcells, or by introducing an agent into target cells that causes,increases, and/or upregulates expression of SDF-1 (i.e., SDF-1 agent).The SDF-1 protein expressed in the target cells can be an expressionproduct of a genetically modified cell. The target cells can includecells within or about the periphery of the wound or ex vivo cells thatare biocompatible with tissue being treated. The biocompatible cells canalso include autologous cells that are harvested from the subject beingtreated and/or biocompatible allogeneic or syngeneic cells, such asautologous, allogeneic, or syngeneic stem cells (e.g., mesenchymal stemcells), progenitor cells (e.g., multipotent adult progenitor cells)and/or other cells that are further differentiated and are biocompatiblewith the tissue being treated. The cells can include cells that areprovided in skin grafts, bone grafts, engineered tissue, and othertissue replacement therapies that are used to treat wounds.

The agent can comprise natural or synthetic nucleic acids, according topresent invention and described above, that are incorporated intorecombinant nucleic acid constructs, typically DNA constructs, capableof introduction into and replication in the cell. Such a construct caninclude a replication system and sequences that are capable oftranscription and translation of a polypeptide-encoding sequence in agiven target cell.

Other agents can also be introduced into the cells to promote expressionof SDF-1 from the cells. For example, agents that increase thetranscription of a gene encoding SDF-1, increase the translation of anmRNA encoding SDF-1, and/or those that decrease the degradation of anmRNA encoding SDF-1 could be used to increase SDF-1 protein levels.Increasing the rate of transcription from a gene within a cell can beaccomplished by introducing an exogenous promoter upstream of the geneencoding SDF-1. Enhancer elements, which facilitate expression of aheterologous gene, may also be employed.

Other agents can further include other proteins, chemokines, andcytokines, that when administered to the target cells can upregulateexpression SDF-1 form the target cells. Such agents can include, forexample: insulin-like growth factor (IGF)-1, which was shown toupregulate expression of SDF-1 when administered to mesenchymal stemcells (MSCs) (Circ. Res. 2008, November 21; 103(11):1300-98); sonichedgehog (Shh), which was shown to upregulate expression of SDF-1 whenadministered to adult fibroblasts (Nature Medicine, Volume 11, Number11, November 23); transforming growth factor .beta. (TGF-.beta.); whichwas shown to upregulate expression of SDF-1 when administered to humanperitoneal mesothelial cells (HPMCs); IL-1.beta., PDG-BF, VEGF,TNF-.alpha., and PTH, which are shown to upregulate expression of SDF-1,when administered to primary human osteoblasts (HOBS) mixed marrowstromal cells (BMSCs), and human osteoblast-like cell lines (Bone, 2006,April; 38(4): 497-508); thymosin .beta.4, which was shown to upregulateexpression when administered to bone marrow cells (BMCs) (Curr. Pharm.Des. 2007; 13(31):3245-51; and hypoxia inducible factor 1.alpha.(HIF-1), which was shown to upregulate expression of SDF-1 whenadministered to bone marrow derived progenitor cells (Cardiovasc. Res.2008, E. Pub.). These agents can be used to treat specific wounds orinjuries where such cells capable of upregulating expression of SDF-1with respect to the specific cytokine are present or administered.

One method of introducing the agent into a target cell involves usinggene therapy. Gene therapy in accordance with the present invention canbe used to express SDF-1 protein from a target cell in vivo or in vitro.

In an aspect of the invention, the gene therapy can use a vectorincluding a nucleotide encoding an SDF-1 protein. A “vector” (sometimesreferred to as gene delivery or gene transfer “vehicle”) refers to amacromolecule or complex of molecules comprising a polynucleotide to bedelivered to a target cell, either in vitro or in vivo. Thepolynucleotide to be delivered may comprise a coding sequence ofinterest in gene therapy. Vectors include, for example, viral vectors(such as adenoviruses (‘Ad’), adeno-associated viruses (AAV), andretroviruses), liposomes and other lipid-containing complexes, and othermacromolecular complexes capable of mediating delivery of apolynucleotide to a target cell.

Vectors can also comprise other components or functionalities thatfurther modulate gene delivery and/or gene expression, or that otherwiseprovide beneficial properties to the targeted cells. Such othercomponents include, for example, components that influence binding ortargeting to cells (including components that mediate cell-type ortissue-specific binding); components that influence uptake of the vectornucleic acid by the cell; components that influence localization of thepolynucleotide within the cell after uptake (such as agents mediatingnuclear localization); and components that influence expression of thepolynucleotide. Such components also might include markers, such asdetectable and/or selectable markers that can be used to detect orselect for cells that have taken up and are expressing the nucleic aciddelivered by the vector. Such components can be provided as a naturalfeature of the vector (such as the use of certain viral vectors whichhave components or functionalities mediating binding and uptake), orvectors can be modified to provide such functionalities.

Selectable markers can be positive, negative or bifunctional. Positiveselectable markers allow selection for cells carrying the marker,whereas negative selectable markers allow cells carrying the marker tobe selectively eliminated. A variety of such marker genes have beendescribed, including bifunctional (i.e. positive/negative) markers (see,e.g., Lupton, S., WO 92/08796, published May 29, 1992; and Lupton, S.,WO 94/28143, published Dec. 8, 1994). Such marker genes can provide anadded measure of control that can be advantageous in gene therapycontexts. A large variety of such vectors are known in the art and aregenerally available.

Vectors for use in the present invention include viral vectors, lipidbased vectors and other non-viral vectors that are capable of deliveringa nucleotide according to the present invention to the target cells. Thevector can be a targeted vector, especially a targeted vector thatpreferentially binds to cells of proximate the wound. Viral vectors foruse in the invention can include those that exhibit low toxicity to atarget cell and induce production of therapeutically useful quantitiesof SDF-1 protein in a tissue-specific manner.

Examples of viral vectors are those derived from adenovirus (Ad) oradeno-associated virus (AAV). Both human and non-human viral vectors canbe used and the recombinant viral vector can be replication-defective inhumans. Where the vector is an adenovirus, the vector can comprise apolynucleotide having a promoter operably linked to a gene encoding theSDF-1 protein and is replication-defective in humans.

Other viral vectors that can be use in accordance with the presentinvention include herpes simplex virus (HSV)-based vectors. HSV vectorsdeleted of one or more immediate early genes (IE) are advantageousbecause they are generally non-cytotoxic, persist in a state similar tolatency in the target cell, and afford efficient target celltransduction. Recombinant HSV vectors can incorporate approximately 30kb of heterologous nucleic acid.

Retroviruses, such as C-type retroviruses and lentiviruses, might alsobe used in the invention. For example, retroviral vectors may be basedon murine leukemia virus (MLV). See, e.g., Hu and Pathak, Pharmacol.Rev. 52:493-511, 2000 and Fong et al., Crit. Rev. Ther. Drug CarrierSyst. 17:1-60, 2000. MLV-based vectors may contain up to 8 kb ofheterologous (therapeutic) DNA in place of the viral genes. Theheterologous DNA may include a tissue-specific promoter and an SDF-1nucleic acid. In methods of delivery to cells proximate the wound, itmay also encode a ligand to a tissue specific receptor.

Additional retroviral vectors that might be used arereplication-defective lentivirus-based vectors, including humanimmunodeficiency (HIV)-based vectors. See, e.g., Vigna and Naldini, J.Gene Med. 5:308-316, 2000 and Miyoshi et al., J. Virol. 72:8150-8157,1998. Lentiviral vectors are advantageous in that they are capable ofinfecting both actively dividing and non-dividing cells. They are alsohighly efficient at transducing human epithelial cells.

Lentiviral vectors for use in the invention may be derived from humanand non-human (including SIV) lentiviruses. Examples of lentiviralvectors include nucleic acid sequences required for vector propagationas well as a tissue-specific promoter operably linked to a SDF-1 gene.These former may include the viral LTRs, a primer binding site, apolypurine tract, att sites, and an encapsidation site.

A lentiviral vector may be packaged into any suitable lentiviral capsid.The substitution of one particle protein with another from a differentvirus is referred to as “pseudotyping”. The vector capsid may containviral envelope proteins from other viruses, including murine leukemiavirus (MLV) or vesicular stomatitis virus (VSV). The use of the VSVG-protein yields a high vector titer and results in greater stability ofthe vector virus particles.

Alphavirus-based vectors, such as those made from semliki forest virus(SFV) and sindbis virus (SIN), might also be used in the invention. Useof alphaviruses is described in Lundstrom, K., Intervirology 43:247-257,2000 and Perri et al., Journal of Virology 74:9802-9807, 2000.

Recombinant, replication-defective alphavirus vectors are advantageousbecause they are capable of high-level heterologous (therapeutic) geneexpression, and can infect a wide target cell range. Alphavirusreplicons may be targeted to specific cell types by displaying on theirvirion surface a functional heterologous ligand or binding domain thatwould allow selective binding to target cells expressing a cognatebinding partner. Alphavirus replicons may establish latency, andtherefore long-term heterologous nucleic acid expression in a targetcell. The replicons may also exhibit transient heterologous nucleic acidexpression in the target cell.

In many of the viral vectors compatible with methods of the invention,more than one promoter can be included in the vector to allow more thanone heterologous gene to be expressed by the vector. Further, the vectorcan comprise a sequence which encodes a signal peptide or other moietywhich facilitates the secretion of a SDF-1 gene product from the targetcell.

To combine advantageous properties of two viral vector systems, hybridviral vectors may be used to deliver a SDF-1 nucleic acid to a targettissue. Standard techniques for the construction of hybrid vectors arewell-known to those skilled in the art. Such techniques can be found,for example, in Sambrook, et al., In Molecular Cloning: A laboratorymanual. Cold Spring Harbor, N.Y. or any number of laboratory manualsthat discuss recombinant DNA technology. Double-stranded AAV genomes inadenoviral capsids containing a combination of AAV and adenoviral ITRsmay be used to transduce cells. In another variation, an AAV vector maybe placed into a “gutless”, “helper-dependent” or “high-capacity”adenoviral vector. Adenovirus/AAV hybrid vectors are discussed in Lieberet al., J. Virol. 73:9314-9324, 1999. Retrovirus/adenovirus hybridvectors are discussed in Zheng et al., Nature Biotechnol. 18:176-186,2000. Retroviral genomes contained within an adenovirus may integratewithin the target cell genome and effect stable SDF-1 gene expression.

Other nucleotide sequence elements which facilitate expression of theSDF-1 gene and cloning of the vector are further contemplated. Forexample, the presence of enhancers upstream of the promoter orterminators downstream of the coding region, for example, can facilitateexpression.

In accordance with another aspect of the present invention, atissue-specific promoter, can be fused to a SDF-1 gene. By fusing suchtissue specific promoter within the adenoviral construct, transgeneexpression is limited to a particular tissue. The efficacy of geneexpression and degree of specificity provided by tissue specificpromoters can be determined, using the recombinant adenoviral system ofthe present invention.

In addition to viral vector-based methods, non-viral methods may also beused to introduce a SDF-1 nucleic acid into a target cell. A review ofnon-viral methods of gene delivery is provided in Nishikawa and Huang,Human Gene Ther. 12:861-870, 2001. An example of a non-viral genedelivery method according to the invention employs plasmid DNA tointroduce a SDF-1 nucleic acid into a cell. Plasmid-based gene deliverymethods are generally known in the art.

Synthetic gene transfer molecules can be designed to form multimolecularaggregates with plasmid DNA. These aggregates can be designed to bind toa target cell. Cationic amphiphiles, including lipopolyamines andcationic lipids, may be used to provide receptor-independent SDF-1nucleic acid transfer into target cells (e.g., cardiomyocytes). Inaddition, preformed cationic liposomes or cationic lipids may be mixedwith plasmid DNA to generate cell-transfecting complexes. Methodsinvolving cationic lipid formulations are reviewed in Feigner et al.,Ann N.Y. Acad. Sci. 772:126-139, 1995 and Lasic and Templeton, Adv. DrugDelivery Rev. 20:221-266, 1996. For gene delivery, DNA may also becoupled to an amphipathic cationic peptide (Fominaya et al., J. GeneMed. 2:455-464, 2000).

Methods that involve both viral and non-viral based components may beused according to the invention. For example, an Epstein Barr virus(EBV)-based plasmid for therapeutic gene delivery is described in Cui etal., Gene Therapy 8:1508-1513, 2001. Additionally, a method involving aDNA/ligand/polycationic adjunct coupled to an adenovirus is described inCuriel, D. T., Nat. Immun. 13:141-164, 1994.

Additionally, the SDF-1 nucleic acid can be introduced into the targetcell by transfecting the target cells using electroporation techniques.Electroporation techniques are well known and can be used to facilitatetransfection of cells using plasmid DNA.

Vectors that encode the expression of SDF-1 can be delivered to thetarget cell in the form of an injectable preparation containingpharmaceutically acceptable carrier, such as saline, as necessary. Otherpharmaceutical carriers, formulations and dosages can also be used inaccordance with the present invention.

Where the target cell comprises a cell proximate the wound beingtreated, the vector can be delivered by direct injection at an amountsufficient for the SDF-1 protein to be expressed to a degree whichallows for highly effective therapy. By injecting the vector directlyinto or about the periphery of the wound, it is possible to target thevector transfection rather effectively, and to minimize loss of therecombinant vectors. This type of injection enables local transfectionof a desired number of cells, especially about the wound, therebymaximizing therapeutic efficacy of gene transfer, and minimizing thepossibility of an inflammatory response to viral proteins.

Where the target cell is a cultured cell that is later transplanted intowound (e.g., tissue graft), the vectors can be delivered by directinjection into the culture medium. A SDF-1 nucleic acid transfected intocells may be operably linked to a regulatory sequence.

The transfected target cells can then be transplanted to the wound bywell known transplantation techniques, such as graft transplantation. Byfirst transfecting the target cells in vitro and then transplanting thetransfected target cells to the wound, the possibility of inflammatoryresponse in the tissue proximate the wound is minimized compared todirect injection of the vector into cells proximate the wound.

SDF-1 can be expressed for any suitable length of time within the targetcell, including transient expression and stable, long-term expression.In one aspect of the invention, the SDF-1 nucleic acid will be expressedin therapeutic amounts for a defined length of time effective tomitigate apoptosis in the cells proximate the wound and/or to promotestem cell or progenitor cell homing to the wound. This amount of timecan be that amount effect to promote healing of the wound, accelerateclosure of the wound, and/or inhibit scar formation.

A therapeutic amount is an amount, which is capable of producing amedically desirable result in a treated animal or human. As is wellknown in the medical arts, dosage for any one animal or human depends onmany factors, including the subject's size, body surface area, age, theparticular composition to be administered, sex, time and route ofadministration, general health, and other drugs being administeredconcurrently. Specific dosages of proteins and nucleic acids can bedetermined readily determined by one skilled in the art using theexperimental methods described below.

The SDF-1 protein or agent, which causes, increases, and/or upregulatesexpression of SDF-1 from target cells, can be administered to the cellsof the wound, cells proximate wound, or cells administered to the wound(e.g., MSCs transfected to express SDF-1) neat or in a pharmaceuticalcomposition. The pharmaceutical composition can provide localizedrelease of the SDF-1 or agent to the cells proximate the wound, cellsbeing treated, or cells administered to the wound. Pharmaceuticalcompositions in accordance with the invention will generally include anamount of SDF-1 or agent admixed with an acceptable pharmaceuticaldiluent or excipient, such as a sterile aqueous solution, to give arange of final concentrations, depending on the intended use. Thetechniques of preparation are generally well known in the art asexemplified by Remington's Pharmaceutical Sciences, 16th Ed. MackPublishing Company, 1980, incorporated herein by reference. Moreover,for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards.

The pharmaceutical composition can be in a unit dosage injectable form(e.g., solution, suspension, and/or emulsion). Examples ofpharmaceutical formulations that can be used for injection includesterile aqueous solutions or dispersions and sterile powders forreconstitution into sterile injectable solutions or dispersions. Thecarrier can be a solvent or dispersing medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, liquidpolyethylene glycol, and the like), suitable mixtures thereof andvegetable oils.

Proper fluidity can be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for compound compositions

Additionally, various additives which enhance the stability, sterility,and isotonicity of the compositions, including antimicrobialpreservatives, antioxidants, chelating agents, and buffers, can beadded. Prevention of the action of microorganisms can be ensured byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. In many cases, it willbe desirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tothe present invention, however, any vehicle, diluent, or additive usedwould have to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various amounts of the otheringredients, as desired.

Pharmaceutical “slow release” capsules or “sustained release”compositions or preparations may be used and are generally applicable.Slow release formulations are generally designed to give a constant druglevel over an extended period and may be used to deliver the SDF-1 oragent. The slow release formulations are typically implanted in thevicinity of the wound site, for example, at the site of cell expressingCXCR4 and/or CXCR7 in or about the wound.

Examples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymers containing the SDF-1 or agent,which matrices are in the form of shaped articles, e.g., films ormicrocapsule. Examples of sustained-release matrices include polyesters;hydrogels, for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol); polylactides, e.g., U.S. Pat. No. 3,773,919;copolymers of L-glutamic acid and γ ethyl-L-glutamate; non-degradableethylene-vinyl acetate; degradable lactic acid-glycolic acid copolymers,such as the LUPRON DEPOT (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate); andpoly-D-(−)-3-hydroxybutyric acid.

While polymers, such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated, SDF-1 orthe agent can remain in the body for a long time, and may denature oraggregate as a result of exposure to moisture at 37° C., thus reducingbiological activity and/or changing immunogenicity. Rational strategiesare available for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism involves intermolecular S—S bondformation through thio-disulfide interchange, stabilization is achievedby modifying sulfhydryl residues, lyophilizing from acidic solutions,controlling moisture content, using appropriate additives, developingspecific polymer matrix compositions, and the like.

In certain embodiments, liposomes and/or nanoparticles may also beemployed with the SDF-1 or agent. The formation and use of liposomes isgenerally known to those of skill in the art, as summarized below.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs)). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 .ANG., containing an aqueous solution in the core.

Phospholipids can form a variety of structures other than liposomes whendispersed in water, depending on the molar ratio of lipid to water. Atlow ratios, the liposome is the preferred structure. The physicalcharacteristics of liposomes depend on pH, ionic strength and thepresence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

Liposomes interact with cells via four different mechanisms: Endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. Varying the liposome formulation can alter whichmechanism is operative, although more than one may operate at the sametime.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) should be designed usingpolymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and such particles may beare easily made.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers can be in anysuitable form (e.g., solids, liquids, gels, etc.). A solid carrier canbe one or more substances which may also act as diluents, flavoringagents, binders, preservatives, and/or an encapsulating material.

In another aspect of the present invention, the SDF-1 or SDF-1 agent canbe formulated for topical administration to treat surface wounds.Topical formulations include those for delivery via the mouth (buccal)and to the skin such that at least one layer of skin (i.e., theepidermis, dermis, and/or subcutaneous layer) is contacted with SDF-1 oragent. Topical delivery systems may be used to administer topicalformulations of the present invention.

Formulations for topical administration to the skin can includeointments, creams, gels, and pastes comprising SDF-1 or SDF-1 agent tobe administered in a pharmaceutically acceptable carrier. Topicalformulations can be prepared using oleaginous or water-soluble ointmentbases, as is well known to those in the art. For example, theseformulations may include vegetable oils, animal fats, and morepreferably semisolid hydrocarbons obtained from petroleum. Particularcomponents used may include white ointment, yellow ointment, cetylesters wax, oleic acid, olive oil, paraffin, petrolatum, whitepetrolatum, spermaceti, starch glycerite, white wax, yellow wax,lanolin, anhydrous lanolin, and glyceryl monostearate. Variouswater-soluble ointment bases may also be used including, for example,glycol ethers and derivatives, polyethylene glycols, polyoxyl 40stearate, and polysorbates.

In another aspect of the invention, SDF-1 or agent can be provided inand/or on a substrate, solid support, and/or wound dressing for deliveryof the SDF-1 or agent to the wound. As used herein, the term“substrate,” or “solid support” and “wound dressing” refer broadly toany substrate when prepared for, and applied to, a wound for protection,absorbance, drainage, etc. The present invention may include any one ofthe numerous types of substrates and/or backings that are commerciallyavailable, including films (e.g., polyurethane films), hydrocolloids(hydrophilic colloidal particles bound to polyurethane foam), hydrogels(cross-linked polymers containing about at least 60% water), foams(hydrophilic or hydrophobic), calcium alginates (non-woven composites offibers from calcium alginate), and cellophane (cellulose with aplasticizer). The shape and size of a wound may be determined and thewound dressing customized for the exact site based on the measurementsprovided for the wound. As wound sites can vary in terms of mechanicalstrength, thickness, sensitivity, etc., the substrate can be molded tospecifically address the mechanical and/or other needs of the site. Forexample, the thickness of the substrate may be minimized for locationsthat are highly innervated, e.g., the fingertips. Other wound sites,e.g., fingers, ankles, knees, elbows and the like, may be exposed tohigher mechanical stress and require multiple layers of the substrate.

In one example, the substrate can be a bioresorbable implant thatincludes a polymeric matrix and the SDF-1 or agent dispersed in thematrix. The polymeric matrix may be in the form of a membrane, sponge,gel, or any other desirable configuration. The polymeric matrix can beformed from biodegradable polymer. It will be appreciated, however, thatthe polymeric matrix may additionally comprise an inorganic or organiccomposite. The polymeric matrix can comprise any one or combination ofknown materials including, for example, chitosan, poly(ethylene oxide),poly (lactic acid), poly(acrylic acid), poly(vinyl alcohol),poly(urethane), poly(N-isopropyl acrylamide), poly(vinyl pyrrolidone)(PVP), poly (methacrylic acid), poly(p-styrene carboxylic acid),poly(p-styrenesulfonic acid), poly(vinylsulfonicacid),poly(ethyleneimine), poly(vinylamine), poly(anhydride), poly(L-lysine),poly(L-glutamic acid), poly(gamma-glutamic acid), poly(caprolactone),polylactide, poly(ethylene), poly(propylene), poly(glycolide),poly(lactide-co-glycolide), poly(amide), poly(hydroxylacid),poly(sulfone), poly(amine), poly(saccharide), poly(HEMA),poly(anhydride), collagen, gelatin, glycosaminoglycans (GAG), poly(hyaluronic acid), poly(sodium alginate), alginate, hyaluronan, agarose,polyhydroxybutyrate (PHB), and the like.

It will be appreciated that one having ordinary skill in the art maycreate a polymeric matrix of any desirable configuration, structure, ordensity. By varying polymer concentration, solvent concentration,heating temperature, reaction time, and other parameters, for example,one having ordinary skill in the art can create a polymeric matrix withany desired physical characteristic(s). For example, the polymericmatrix may be formed into a sponge-like structure of various densities.The polymeric matrix may also be formed into a membrane or sheet whichcould then be wrapped around or otherwise shaped to a wound. Thepolymeric matrix may also be configured as a gel, mesh, plate, screw,plug, or rod. Any conceivable shape or form of the polymeric matrix iswithin the scope of the present invention. In an example of the presentinvention, the polymeric matrix can comprise a alginate matrix.

In another aspect of the present invention, at least one progenitor cellcan be provided in the polymeric matrix. Examples progenitor cells canbe selected from, but not restricted to, totipotent stem cell,pluripotent stem cell, multipotent stem cell, mesenchymal stem cell,neuronal stem cell, hematopoietic stem cell, pancreatic stem cell,cardiac stem cell, embryonic stem cell, embryonic germ cell, neuralcrest stem cell, kidney stem cell, hepatic stem cell, lung stem cell,hemangioblast cell, and endothelial progenitor cell. Additional examplesof progenitor cells can be selected from, but not restricted to,de-differentiated chondrogenic cells, myogenic cells, osteogenic cells,tendogenic cells, ligamentogenic cells, adipogenic cells, anddermatogenic cells.

The polymeric matrix of the present invention may be seeded with atleast one progenitor cell and the SDF-1 or agent. The SDF-1 or agent canbe dispersed in matrix and/or expressed from the seeded progenitor cell.Progenitor cells can include autologous cells; however, it will beappreciated that xenogeneic, allogeneic, or syngeneic cells may also beused. Where the cells are not autologous, it may be desirable toadminister immunosuppressive agents in order to minimizeimmunorejection. The progenitor cells employed may be primary cells,explants, or cell lines, and may be dividing or non-dividing cells.Progenitor cells may be expanded ex vivo prior to introduction into thepolymeric matrix. Autologous cells are preferably expanded in this wayif a sufficient number of viable cells cannot be harvested from thehost.

The SDF-1 or SDF-1 agent can also be provided in or on a surface of amedical device used to treat an internal and/or external wound. Themedical device can comprise any instrument, implement, machine,contrivance, implant, or other similar or related article, including acomponent or part, or accessory, which is, for example, recognized inthe official U.S. National Formulary, the U.S. Pharmacopoeia, or anysupplement thereof; is intended for use in the diagnosis of disease orother conditions, or in the cure, mitigation, treatment, or preventionof disease, in humans or in other animals; or, is intended to affect thestructure or any function of the body of humans or other animals, andwhich does not achieve any of its primary intended purposes throughchemical action within or on the body of man or other animals, and whichis not dependent upon being metabolized for the achievement of any ofits primary intended purposes.

The medical device can include, for example, endovascular medicaldevices, such as intracoronary medical devices. Examples ofintracoronary medical devices can include stents, drug deliverycatheters, grafts, and drug delivery balloons utilized in thevasculature of a subject. Where the medical device comprises a stent,the stent may include peripheral stents, peripheral coronary stents,degradable coronary stents, non-degradable coronary stents,self-expanding stents, balloon-expanded stents, and esophageal stents.The medical device may also include arterio-venous grafts, by-passgrafts, penile implants, vascular implants and grafts, intravenouscatheters, small diameter grafts, artificial lung catheters,electrophysiology catheters, bone pins, suture anchors, blood pressureand stent graft catheters, breast implants, benign prostatic hyperplasiaand prostate cancer implants, bone repair/augmentation devices, breastimplants, orthopedic joint implants, dental implants, implanted druginfusion tubes, oncological implants, pain management implants,neurological catheters, central venous access catheters, catheter cuff,vascular access catheters, urological catheters/implants, atherectomycatheters, clot extraction catheters, PTA catheters, PTCA catheters,stylets (vascular and non-vascular), drug infusion catheters,angiographic catheters, hemodialysis catheters, neurovascular ballooncatheters, thoracic cavity suction drainage catheters, electrophysiologycatheters, stroke therapy catheters, abscess drainage catheters, biliarydrainage products, dialysis catheters, central venous access catheters,and parental feeding catheters.

The medical device may additionally include either implantablepacemakers or defibrillators, vascular grafts, sphincter devices,urethral devices, bladder devices, renal devices, gastroenteral andanastomotic devices, vertebral disks, hemostatic barriers, clamps,surgical staples/sutures/screws/plates/wires/clips, glucose sensors,blood oxygenator tubing, blood oxygenator membranes, blood bags, birthcontrol/IUDs and associated pregnancy control devices, cartilage repairdevices, orthopedic fracture repairs, tissue scaffolds, CSF shunts,dental fracture repair devices, intravitreal drug delivery devices,nerve regeneration conduits, electrostimulation leads, spinal/orthopedicrepair devices, wound dressings, embolic protection filters, abdominalaortic aneurysm grafts and devices, neuroaneurysm treatment coils,hemodialysis devices, uterine bleeding patches, anastomotic closures,aneurysm exclusion devices, neuropatches, vena cava filters, urinarydilators, endoscopic surgical and wound drainings, surgical tissueextractors, transition sheaths and dilators, coronary and peripheralguidewires, circulatory support systems, tympanostomy vent tubes,cerebro-spinal fluid shunts, defibrillator leads, percutaneous closuredevices, drainage tubes, bronchial tubes, vascular coils, vascularprotection devices, vascular intervention devices including vascularfilters and distal support devices and emboli filter/entrapment aids, AVaccess grafts, surgical tampons, cardiac valves, and tissue engineeredconstructs, such as bone grafts and skin grafts.

The following examples are for the purpose of illustration only and arenot intended to limit the scope of the claims, which are appendedhereto.

Example 1 Stromal Cell-Derived Factor-1 Release in Alginate Scaffolds:Characterization and Ability to Accelerate Wound Healing

We hypothesized that a slow-release delivery of either SDF-1 protein orplasmid would increase its effectiveness on wound healing. Therefore, weemployed a clinically-relevant delivery system, an alginate scaffold, todeliver SDF-1 over time to a porcine acute surgical wound model. Wecharacterize SDF-1 delivery using alginate scaffolds in vitro, anddemonstrated the potential for therapeutic benefit in vivo by using thescaffolds to deliver SDF-1 protein and plasmid to acute surgical wounds.

Preparation of Scaffolds for In Vivo Application

For the in vivo application, custom 1 cm×6 cm alginate scaffolds wereproduced by the same process described above. Scaffolds were then loadedwith SDF-1 plasmid (n=6), SDF-1 protein (n=10), or phosphate bufferedsaline (PBS) (n=4) by the process described below.

For the SDF-1 plasmid scaffolds, a plasmid was created by inserting thegene encoding human SDF-1 in a pcDNA3.1 backbone (InvitrogenCorporation, Carlsbad, Calif.). A loading solution was prepared bymixing 3.5 mg of the SDF-1 plasmid in 2.33 ml PBS to create a 1.5 mg/mlsolution. On each scaffold, the loading solution was pipetted understerile conditions onto the scaffold in six 60 μl drops (360 μl total)equally spaced so that each drop covered a 1 cm×1 cm area of thescaffold.

For the SDF-1 protein scaffolds, a loading solution was prepared bymixing 10 μg of carrier-free SDF-1 protein (R&D systems, Minneapolis,Minn.) with 5 mL PBS and 3 ml of 1000 IU/ml injection heparin (BaxterHealthcare Corporation, Deerfield, Ill.) to create a 1.5 μg/ml solution.On each scaffold, the loading solution was pipetted under sterileconditions onto the scaffold in six equally spaced 60 μl drops.

The PBS scaffolds served as a negative control. The loading solution wasprepared by mixing 1.35 mL PBS and 0.45 ml of 1000 IU/ml injectionheparin. The loading solution was pipetted under sterile conditions ontothe scaffold in six equally spaced 60 μl drops.

All loaded scaffolds were stored at 4° C. for 12 hours prior to applyingthem to the wounds.

Porcine Surgical Wound Healing Model and Ante-Mortem Follow-Up

In 2 Domestic Yorkshire pigs, general anesthesia was induced. A cuffedendotracheal tube was placed and general anesthesia was maintained withisoflurane delivered in oxygen through a rebreathing system withventilator assist. A standard model of acute surgical wounds was used.Each animal received twelve (12) 5 cm full thickness incisions (six oneach side of the spine) spaced approximately 7.5 cm apart. Each incisionwas made perpendicular to the spine, starting 7.5 cm from the spine andcutting toward the abdomen. Gauze was placed in the incision until thebleeding stopped. The gauze was removed, and the incision was suturedclosed.

Following wound closure, the scaffold was placed next to the wound andphotographed (FIG. 1). On each pig, the scaffold placement order wasrandomized with the following distribution:

-   -   SDF-1 protein scaffold (n=5)    -   SDF-1 plasmid scaffold (n=3)    -   PBS scaffold (control, n=2)    -   No scaffold (sham, n=2)

The scaffold was placed over the wound (except in the sham group), andeach wound was dressed with a Tegaderm™ patch.

To determine the effect of SDF-1 on the rate of wound healing, woundlength was measured by the same veterinarian at day 0 (prior to scaffoldplacement) and prior to sacrifice. Wound length was converted to PercentHealing by the following relationship:

(Initial wound length-final wound length)/initial wound length*100%

To monitor both the acute and chronic effects of SDF-1 on wound healing,the acute effects were evaluated in the first pig, which was sacrificedat 4 days, and the chronic effects in the second which was sacrificed at9 days.

Post-Mortem Follow-Up

Following sacrifice, one section from the middle of each wound site wasexcised for histopathological and immunohistochemical analysis. Standardhematoxylin and eosin (H&E) stain was used to assess extent offibroplasia, inflammation, and necrosis at day 4 and necrosis, fibrosis,and granulomatous inflammation at day 9. Each parameter was graded on aqualitative scale by a histopathologist blinded to randomization aseither: none (not present), minimal, mild, moderate, or severe.Immunohistochemical staining was performed on the same tissue section.The effect of SDF-1 on fibroblast infiltration into the wound wasdetected by vimentin staining. The effect on blood vessel formation wasdetermined by CD31 and the presence of smooth muscle was detected bysmooth muscle actin staining. The amount of each stain per sample wasgraded by the same pathologist using the same qualitative scale as above(minimal severe).

The impact of an SDF-1-releasing scaffold on wound healing is also shownin FIGS. 1 and 2. FIG. 1 shows representative examples of wounds treatedwith control (PBS) scaffold, SDF-1 protein scaffold, and SDF-1 plasmidscaffold at day 0 (top panel) and day 9 (bottom panel). Allfull-incision wounds (middle) have a length 5.0±0.1 cm.

At day 9, the wound treated with the control scaffold is still apparent,and has a Percent Healed of 0%. In contrast, both the SDF-1 protein andSDF-1 plasmid treated wounds are no longer visible at day 9, and bothhave a Percent Healed of 100%.

FIG. 2 summarizes the percent healing data for all treated wounds. Day 4data is from the first pig, and Day 9 data is from the second pig. AtDay 9, the wounds treated with either the SDF-1 plasmid or proteinscaffolds (solid markers and lines) have healed to a greater extent thanthe control or sham groups (open markers and dotted lines). Notably, 1of 3 SDF-1 plasmid treated wounds and 2 of 5 SDF-1 protein treatedwounds are 100% healed at 9 days; whereas, no control or sham wound aregreater than 20% healed at 9 days.

We investigated the impact of SDF-1 on fibroblast infiltration, newblood vessel formation, and smooth muscle using immunohistochemicalstaining for vimentin, CD31, and smooth muscle actin, respectively.There are no substantial differences in amount of any of the stainsbetween groups. H & E analysis showed a slight decrease in fibrosis inthe SDF-1 protein and plasmid treated wounds compared to control orsham, with all other parameters being similar. The results are shownbelow in the following tables.

The results are shown the table below.

Wound Healing H/E data - Day 9 # wounds with fibrosis Sham (no patch) 21 1 0 2 (of 2) 50% Control (saline patch) 2 1 0 1 2 (of 2) 50% SDF1Protein Patch 5 4 1 0 5 (of 5) 80% SDF1 Plasmid Patch 3 3 0 0 3 (of 3)100%  # wounds with granulomatous inflammation Sham (no patch) 2 0 0 0(of 2) 50% Control (saline patch) 2 0 1 1 (of 2) 50% SDF1 Protein Patch5 1 0 1 (of 5) 80% SDF1 Plasmid Patch 3 0 0 0 (of 3) 100%  # of woundswith necrosis Sham (no patch) 2 1 0 1 (of 2) Control (saline patch) 2 00 0 (of 2) SDF1 Protein Patch 5 1 1 2 (of 5) SDF1 Plasmid Patch 3 1 0 1(of 3) # of wounds with sub-acute inflammation Sham (no patch) 2 1 1 0 2(of 2) Control (saline patch) 2 0 0 0 0 (of 2) SDF1 Protein Patch 5 0 11 2 (of 5) SDF1 Plasmid Patch 3 1 0 1 2 (of 3)

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All patents, patentapplications and publications cited herein are incorporated by referencein their entirety.

What is claimed:
 1. A method for inhibiting and/or mitigating formationof scar tissue in a wound of the skin, comprising increasing theconcentration of SDF-1 in, or proximate to, the wound by administeringto said wound and/or an area proximate the wound a therapeuticallyeffective amount of an SDF-1 expression vector.
 2. The method accordingto claim 1, wherein said wound of the skin is an acute wound selectedfrom a thermal burn, a chemical burn, a radiation burn, a burn caused byexcess exposure to ultraviolet radiation, an injury sustained during amedical procedure, an incision, a trauma-induced injury, a cut or alaceration.
 3. The method according to claim 1, wherein the wound of theskin is a chronic wound selected from a pressure sore, a bedsore, awound related to diabetes or poor circulation, or a wound resulting fromdermatitis or acne.
 4. The method according to claim 1, comprisingadministering an SDF-1 expression vector to said wound and/or an areaproximate the wound.
 5. The method according to claim 4, wherein saidSDF-1 expression vector is a viral vector.
 6. The method according toclaim 4, wherein said SDF-1 expression vector is a non-viral vector. 7.The method according to claim 6, wherein said non-viral vector is a DNAplasmid.
 8. The method according to claim 1, wherein said SDF-1expression vector is administered in the form of a pharmaceuticalcomposition that comprises an SDF-1 expression vector and apharmaceutically acceptable carrier.
 9. The method according to claim 1,wherein said pharmaceutical composition is an injectable formulation.10. The method according to claim 9, wherein said injectable formulationis administered by injection directly into the wound or into an areaproximate the wound.
 11. The method according to claim 1, wherein saidSDF-1 expression vector is administered in the form of a topicalformulation.
 12. The method according to claim 1, wherein said SDF-1expression vector is administered in or on a substrate, solid support orwound dressing.
 13. The method according to claim 12, wherein said SDF-1expression vector is administered in or on a substrate, and thesubstrate is in the form of a bioresorbable implant.
 14. The methodaccording to claim 1, wherein said SDF-1 expression vector isadministered in or on a wound dressing.
 15. The method according toclaim 1, wherein said SDF-1 expression vector is administered to theexternal surface of the wound.
 16. The method according to claim 1,wherein said SDF-1 expression vector is administered as part of asurgical procedure.
 17. The method according to claim 1, wherein saidSDF-1 expression vector is administered within 24 hours of the woundoccurring.
 18. The method according to claim 1, wherein said SDF-1expression vector is administered more than 24 hours after the woundoccurred.